Process for the production of proteins

Details Process for the production of proteins Process for the production of proteins

1999-05-05

2001-09-04

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S320100, C435S069100, C435S069200, C435S069510, C435S069700, C435S254110, C435S254200

Reexamination Certificate

active

06284520

ABSTRACT:

The invention pertains to the field of recombinant DNA technology and concerns a method for the production of proteins with the aid of genetically engineered yeast cells carrying hybrid vectors comprising the genes for said proteins.
BACKGROUND OF THE INVENTION
Although in genetic engineering numerous protein expression systems for prokaryotic and eukaryotic hosts are already known, there is a continuing demand for novel systems which have advantages over the known systems.
Working on the expression of heterologous proteins in the baker yeast
Saccharomyces cerevisiae
, it has been commonly observed, that a high-level expression is dependent on many factors, e.g. plasmid stability, plasmid copy number, promoter strength, translation efficiency, low protein degradation.
In this context, one of the very important requisites is the yeast strain which is used for the production.
Recently, quite a number of heterologous proteins have been expressed in different yeast strains after transformation of yeast cells with suitable expression vectors comprising DNA sequences coding for said proteins, like e.g. &agr;-interferon (Hitzeman et al. Nature (1981), 293, 717-722), tissue-type plasminogen activator (EP-A-143081) or certain desulfatohirudins (EP-A-225633). In many cases, however, the heterologous proteins are not synthesized in pure form, but as a mixture containing partially degraded such as C- or N-terminally shortened proteins. For instance, the expression of human atrial natriuretic peptide (hANP) in yeast resulted in the secretion of two forms of mature hANP differing in their C-terminus (Vlasuk et al. J. Biol. Chem. (1986), 261, 4789-4796). Similar results have been obtained after the expression of epidermal growth factor (EGF) in yeast (George-Nascimento et al. Biochemistry (1988), 27, 797-802) where the secreted expression products were heterologous in that either the last (Arg 53) or the last two amino acids (Leu 52 and Arg 53) were missing and no full-length EGF was produced.
The separation of mixtures containing full-length proteins such as &agr;-interferon, tissue-type plasminogen activator, inhibitors of tissue-type plasminogen activator, or desulfatohirudins as well as partially degraded like C- or N-terminally shortened derivatives thereof into the individual components and the purification of these components to homogeneity, if these derivatives are biologically active at all, is laborious and time-consuming. Considering the incidental expenses there is a need for improved methods which render possible the economic production of homogenous proteins such as desulfatohirudin in yeast. It is an object of the present invention to provide methods for the production of proteins heterologous to yeast in a homogenous form.
Surprisingly it has been found, that the use of
Saccharomyces cerevisiae
strain HT393 for the expression of heterologous proteins leads to increased yield of biologically active and undegraded form of the expressed heterologous protein, compared to other
Saccharomyces cerevisiae
strains that are genetically closely related, e.g., to strain cl3-ABYS-86 (DSM 9698) that is genetically closest related.
DESCRIPTION OF THE INVENTION
The present invention concerns a process for the production of a protein heterologous to yeast in a homogenous form characterized in that
Saccharomyces cerevisiae
strain HT393 (DSM 9697) or a derivative thereof is used for the expression of said heterologous protein.
In a preferred embodiment, the present invention relates to an improved process for the production of a protein heterologous to yeast in a homogenous form comprising culturing
Saccharomyces cerevisiae
strain HT393 (DSM 9697) or a derivative thereof that has been transformed with a hybrid vector comprising a DNA sequence coding for said heterologous protein and isolating said heterologous protein.
A derivative of HT393 is a strain that is derived from HT393 and shows the same properties in respect to the production of heterologous proteins. The use of the inventive strains leads, e.g., to an increased yield of a biologically active and undegraded form of an expressed heterologous protein.
This heterologous protein can also be processed further, e.g. glycosylated. Useful proteins are, for example, enzymes that can be used, for the production of nutrients and for performing enzymatic reactions in chemistry, or proteins which are useful and valuable as nutrients or for the treatment of human or animal diseases or for the prevention thereof, for example hormones, polypeptides with immunomodulatory, anti-viral and anti-tumor properties, antibodies, viral antigens, vaccines, clotting factors, enzyme inhibitors, foodstuffs and the like.
Such heterologous structural genes are for example those coding for hormones such as secretin, thymosin, relaxin, calcitonin, luteinizing hormone, parathyroid hormone, adreno adenocorticotropin, melanoycte-stimulating hormone, &bgr;-lipotropin, urogastrone or insulin, growth factors, such as epidermal growth factor, insulin-like growth factor (IGF), e.g. IGF-I and IGF-II, mast cell growth factor, nerve growth factor, glia derived nerve cell growth factor, or transforming growth factor (TGF), such as TGF&agr; or TGF&bgr;, e.g. TGF&bgr;1, &bgr;2 or &bgr;3, growth hormone, such as human or bovine growth hormones, interleukin, such as interleukin-1 or -2, human macrophage migration inhibitory factor (MIF), interferons, such as human &agr;-interferon, for example interferon-&agr;A, &agr;B, &agr;D or &agr;F, &bgr;-interferon, &ggr;-interferon or a hybrid interferon, for example an &agr;A-&agr;D- or an &agr;B-&agr;D-hybrid interferon, especially the hybrid interferon BDBB, inhibitors such as proteinase inhibitors such as &agr;
1
-antitrypsin, SLPI, an inhibitor of the plasminogen activator (PAI-2) and the like, hepatitis virus antigens, such as hepatitis B virus surface or core antigen or hepatitis A virus antigen, or hepatitis nonA-nonB antigen, plasminogen activators, such as tissue plasminogen activator or urokinase, tumor necrosis factor, somatostatin, renin, &bgr;-endorphin, immunoglobulins, such as the light and/or heavy chains of immunoglobulin D, E or G, or human-mouse hybrid immunoglobulins, immunoglobulin binding factors, such as immunoglobulin E binding factor, e.g. sCD23 and the like, calcitonin, human calcitonin-related peptide, blood clotting factors, such as factor IX or VIIIc, erythropoietin, eglin, such as eglin C, desulfatohirudin, such as desulfatohirudin variant HV1, HV2 or PA, human superoxide dismutase, viral thymidin kinase, &bgr;-lactamase, glucose isomerase.
Preferred genes are those coding for a human &agr;-interferon or hybrid interferon, particularly hybrid interferon BDBB and an inhibitor of the plasminogen activator (PAI-2).
In a preferred embodiment of the invention, the expressed heterologous protein is not secreted.
S. cerevisiae
strain HT 393 (E95-1-2A) is obtained from strain cl3-ABYS-86 (DSM 9698) as described in Heinemeyer et al, EMBO J. (1991), 10, 555-562.
The wording derivatives of
S. cerevisiae
strain HT 393 embraces strains that are derived by genetic engineering from HT 393 and are, e.g., strains that are additionally cleared from two-micron (2&mgr;) DNA (cir
0
), have a different mating type (MAT&agr;) and/or different selection marker. Preferred is HT 393 and derivatives thereof that are also deficient for protease A, protease B, carboxypeptidase Y, carboxypeptidase S and proteinase yscE.
Essentially preferred are HT 393 and derivatives thereof that show at least the following genetic characterization MATa, leu2-3, leu2-112, ura3&Dgr;5, prb1-1, cps1-3, prc1-1, pra1-1 and pre1-1.
Expression Cassettes
A suitable expression cassette comprises a promoter operably linked to a DNA sequence coding for the protein and to a DNA sequence containing transcription termination signals.
The expression cassette may additionally comprise a DNA sequence encoding a signal peptide linked in the proper reading frame to the DNA sequence coding for the inventive protein.
In a preferred embodiment, the promoter, the sig

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